Patent Application
20220265822
This application includes an electronic sequence listing in a file named SEQ_Listing.txt created on Nov. 18, 2021 and containing 6.08 KB which is hereby incorporated by reference.
This disclosure relates methods of using a non-fucosylated anti-CD40 antibody for treatment of cancer and chronic infectious diseases.
CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily. It is a single chain type I transmembrane protein with an apparent MW of 50 kDa. CD40 is expressed by some cancer cells, e.g., lymphoma cells and several types of solid tumor cells. CD40 also functions to activate the immune system by facilitating contact-dependent reciprocal interaction between antigen-presenting cells and T cells. Although a number of anti-CD40 antibodies have been tested in clinical trials, to date none have exhibited sufficient activity. The present disclosure solves this and other problems.
This disclosure provides a method of treating cancer, by administering an anti-CD40 antibody to a patient in need of such treatment. The anti-CD40 antibody comprises the heavy chain variable region of SEQ ID NO:1 and the light chain variable region of SEQ ID NO:2, and a human constant region. The constant region has an N-glycoside-linked sugar chain at residue N297 according to the EU index as set forth in Kabat and less than 5% of the N-glycoside-linked sugar chains include a fucose residue, i.e., a fucose bound to the reducing terminal of the sugar chain via an α1,6 bond to N-acetylglucosamine (“GlcNAc”). Administration of the anti-CD40 antibody is at a dose level between 0.1-300 μg/kg (μg antibody per kilogram patient body weight). In one embodiment, the anti-CD40 antibody dose level is between 0.6-150 μg/kg. In another embodiment, the anti-CD40 antibody dose level is between 1.0-100 μg/kg. In another embodiment, the anti-CD40 antibody dose level is between 5-25 μg/kg. In another embodiment, the anti-CD40 antibody dose level is between 8-12 μg/kg. In another embodiment, the anti-CD40 antibody dose level is about 10 μg/kg. In another embodiment, the anti-CD40 antibody the dose level is 10 μg/kg.
In another aspect, this disclosure provides a method of treating cancer, by administering an anti-CD40 antibody to a patient in need of such treatment. The anti-CD40 antibody comprises the heavy chain variable region of SEQ ID NO:1 and the light chain variable region of SEQ ID NO:2, and a human constant region. The constant region has an N-glycoside-linked sugar chain at residue N297 according to the EU index as set forth in Kabat and less than 5% of the N-glycoside-linked sugar chains include a fucose residue, i.e., a fucose bound to the reducing terminal of the sugar chain via an α1,6 bond to N-acetylglucosamine (“GlcNAc”). Administration of the anti-CD40 antibody is at a dose level between 0.1-2000 μg/kg (μg antibody per kilogram patient body weight). In one embodiment, the dose level is between 10-1000 μg/kg. In another embodiment, the dose level is between 50-800 μg/kg. In a further embodiment, the dose level is between 75-600 μg/kg. In another embodiment, the dose level is between 100-500 μg/kg. in further embodiments, the dose level is a range selected from the following: 100-300 μg/kg, 300-500 μg/kg, 500-700 μg/kg, 700-900 μg/kg, and 900-1100 μg/kg. In other embodiments, the dose level is a range selected from the following: 100-150 μg/kg, 150-200 μg/kg, 200-250 μg/kg, 250-300 μg/kg, 300-350 μg/kg, 350-400 μg/kg, 400-450 μg/kg, 450-500 μg/kg, 500-550 μg/kg, 550-600 μg/kg, 600-650 μg/kg, 650-700 μg/kg, 700-750 μg/kg, 750-800 μg/kg, 800-850 μg/kg, 850-900 μg/kg, 900-950 μg/kg, 950-1000 μg/kg, 1000-1050 μg/kg, and 1050-1100 μg/kg. In further embodiments, the dose level is selected from the following: about 60 μg/kg, about 100 μg/kg, about 150 μg/kg, about 200 μg/kg, about 250 μg/kg, about 300 μg/kg, about 350 μg/kg, about 400 μg/kg, about 450 μg/kg, about 500 μg/kg, about 550 μg/kg, about 600 μg/kg, about 650 μg/kg, about 700 μg/kg, about 750 μg/kg, about 800 μg/kg, about 850 μg/kg, about 900 μg/kg, about 950 μg/kg, about 1000-1050 μg/kg, about 1050 μg/kg, and 1110 μg/kg.
In one embodiment, the anti-CD40 antibody is administered every three weeks. In another embodiment the anti-CD40 antibody is administered every six weeks. In another embodiment the anti-CD40 antibody is administered every ten weeks. In another embodiment the anti-CD40 antibody is administered every twelve weeks. In another embodiment the anti-CD40 antibody is administered every fifteen weeks. In another embodiment the anti-CD40 antibody is administered every eighteen weeks.
In another embodiment, the patient has a CD40 positive cancer. In another embodiment, the patient has a CD40 negative cancer. In a further embodiment, the patient has a cancer that is a solid tumor. In yet another embodiment, the patient has a cancer that is a blood cancer. In another embodiment, the cancer is a melanoma, a breast cancer, including metastatic breast cancer, a lung cancer, including a non-small cell lung cancer, or pancreatic cancer.
In a further aspect, this disclosure provides methods of treating cancer by administering to the patient a combination of the anti-CD40 antibody and an antibody that blocks an immune checkpoint. One example of an antibody that blocks an immune checkpoint is an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibody. Examples of anti-CTLA4 antibodies include, e.g., ipilimumab or tremelimumab. Another example of an antibody that blocks an immune checkpoint is an anti-programmed cell death protein 1 (PD1) antibody. Examples of anti-PD1 antibodies include, e.g., nivolumab, pidilizumab, or pembrolizumab. A further example of an antibody that blocks an immune checkpoint is an anti-programmed death-ligand (PD-L1) antibody. Examples of anti-PD-L1 antibodies include, e.g., MEDI4736 and MPDL3280A.
In another embodiment, the patient has a CD40 positive cancer and is treated with a combination of the anti-CD40 antibody and an antibody that blocks an immune checkpoint, e.g., an anti-CTLA4 antbody, an anti-PD1 antibody, or an anti-PD-L1 antibody. In another embodiment, the patient has a CD40 negative cancer and is treated with a combination of the anti-CD40 antibody and an antibody that blocks an immune checkpoint, e.g., an anti-CTLA4 antbody, an anti-PD1 antibody, or an anti-PD-L1 antibody. In a further embodiment, the patient has a cancer that is a solid tumor and is treated with a combination of the anti-CD40 antibody and an antibody that blocks an immune checkpoint, e.g., an anti-CTLA4 antbody, an anti-PD1 antibody, or an anti-PD-L1 antibody. In yet another embodiment, the patient has a cancer that is a blood cancer and is treated with a combination of the anti-CD40 antibody and an antibody that blocks an immune checkpoint, e.g., an anti-CTLA4 antbody, an anti-PD1 antibody, or an anti-PD-L1 antibody. In another embodiment, the cancer is a melanoma, a breast cancer, including metastatic breast cancer, a lung cancer, including a non-small cell lung cancer, or pancreatic cancer, and is treated with a combination of the anti-CD40 antibody and an antibody that blocks an immune checkpoint, e.g., an anti-CTLA4 antbody, an anti-PD1 antibody, or an anti-PD-L1 antibody.
A “polypeptide” or “polypeptide chain” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides.”
A “protein” is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
The terms “amino-terminal” and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
The term “antibody” is used herein to denote immunoglobulin proteins produced by the body in response to the presence of an antigen and that bind to the antigen, as well as antigen-binding fragments and engineered variants thereof. Hence, the term “antibody” includes, for example, intact monoclonal antibodies comprising full-length immunoglobulin heavy and light chains (e.g., antibodies produced using hybridoma technology) and antigen-binding antibody fragments, such as F(ab′)2 and Fab fragments. Genetically engineered intact antibodies and fragments, such as chimeric antibodies, humanized antibodies, single-chain Fv fragments, single-chain antibodies, diabodies, minibodies, linear antibodies, multivalent or multispecific (e.g., bispecific) hybrid antibodies, and the like are also included. Thus, the term “antibody” is used expansively to include any protein that comprises an antigen-binding site of an antibody and is capable of specifically binding to its antigen.
An “antigen-binding site of an antibody” is that portion of an antibody that is sufficient to bind to its antigen. The minimum such region is typically a variable domain or a genetically engineered variant thereof. Single-domain binding sites can be generated from camelid antibodies (see Muyldermans and Lauwereys, J. Mol. Recog. 12:131-140, 1999; Nguyen et al., EMBO J. 19:921-930, 2000) or from VH domains of other species to produce single-domain antibodies (“dAbs”; see Ward et al., Nature 341:544-546, 1989; U.S. Pat. No. 6,248,516 to Winter et al.). In certain variations, an antigen-binding site is a polypeptide region having only 2 complementarity determining regions (CDRs) of a naturally or non-naturally (e.g., mutagenized) occurring heavy chain variable domain or light chain variable domain, or combination thereof (see, e.g., Pessi et al., Nature 362:367-369, 1993; Qiu et al., Nature Biotechnol. 25:921-929, 2007). More commonly, an antigen-binding site of an antibody comprises both a heavy chain variable (VH) domain and a light chain variable (VL) domain that bind to a common epitope. Within the context of the present invention, an antibody may include one or more components in addition to an antigen-binding site, such as, for example, a second antigen-binding site of an antibody (which may bind to the same or a different epitope or to the same or a different antigen), a peptide linker, an immunoglobulin constant region, an immunoglobulin hinge, an amphipathic helix (see Pack and Pluckthun, Biochem. 31:1579-1584, 1992), a non-peptide linker, an oligonucleotide (see Chaudri et al., FEBS Letters 450:23-26, 1999), a cytostatic or cytotoxic drug, and the like, and may be a monomeric or multimeric protein. Examples of molecules comprising an antigen-binding site of an antibody are known in the art and include, for example, Fv, single-chain Fv (scFv), Fab, Fab′, F(ab′)2, F(ab)c, diabodies, dAbs, minibodies, nanobodies, Fab-scFv fusions, bispecific (scFv)4-IgG, and bispecific (scFv)2-Fab. (See, e.g., Hu et al., Cancer Res. 56:3055-3061, 1996; Atwell et al., Molecular Immunology 33:1301-1312, 1996; Carter and Merchant, Curr. Opin. Biotechnol. 8:449-454, 1997; Zuo et al., Protein Engineering 13:361-367, 2000; and Lu et al., J. Immunol. Methods 267:213-226, 2002.)
As used herein, the term “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin gene(s). One form of immunoglobulin constitutes the basic structural unit of native (i.e., natural) antibodies in vertebrates. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions (VL and VH) are together primarily responsible for binding to an antigen, and the constant regions are primarily responsible for the antibody effector functions. Five classes of immunoglobulin protein (IgG, IgA, IgM, IgD, and IgE) have been identified in higher vertebrates. IgG comprises the major class; it normally exists as the second most abundant protein found in plasma. In humans, IgG consists of four subclasses, designated IgG1, IgG2, IgG3, and IgG4. The heavy chain constant regions of the IgG class are identified with the Greek symbol γ. For example, immunoglobulins of the IgG1 subclass contain a γ1 heavy chain constant region. Each immunoglobulin heavy chain possesses a constant region that consists of constant region protein domains (CH1, hinge, CH2, and CH3; IgG3 also contains a CH4 domain) that are essentially invariant for a given subclass in a species. DNA sequences encoding human and non-human immunoglobulin chains are known in the art. (See, e.g., Ellison et al., DNA 1:11-18, 1981; Ellison et al., Nucleic Acids Res. 10:4071-4079, 1982; Kenten et al., Proc. Natl. Acad. Sci. USA 79:6661-6665, 1982; Seno et al., Nuc. Acids Res. 11:719-726, 1983; Riechmann et al., Nature 332:323-327, 1988; Amster et al., Nuc. Acids Res. 8:2055-2065, 1980; Rusconi and Kohler, Nature 314:330-334, 1985; Boss et al., Nuc. Acids Res. 12:3791-3806, 1984; Bothwell et al., Nature 298:380-382, 1982; van der Loo et al., Immunogenetics 42:333-341, 1995; Karlin et al., J. Mol. Evol. 22:195-208, 1985; Kindsvogel et al., DNA 1:335-343, 1982; Breiner et al., Gene 18:165-174, 1982; Kondo et al., Eur. J. Immunol. 23:245-249, 1993; and GenBank Accession No. J00228.) For a review of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol V, Academic Press, Inc., 49-140, 1987; and Padlan, Mol. Immunol. 31:169-217, 1994. The term “immunoglobulin” is used herein for its common meaning, denoting an intact antibody, its component chains, or fragments of chains, depending on the context.
Full-length immunoglobulin “light chains” (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the amino-terminus (encoding about 110 amino acids) and a by a kappa or lambda constant region gene at the carboxyl-terminus. Full-length immunoglobulin “heavy chains” (about 50 Kd or 446 amino acids) are encoded by a variable region gene (encoding about 116 amino acids) and a gamma, mu, alpha, delta, or epsilon constant region gene (encoding about 330 amino acids), the latter defining the antibody's isotype as IgG, IgM, IgA, IgD, or IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. (See generally Fundamental Immunology (Paul, ed., Raven Press, N.Y., 2nd ed. 1989), Ch. 7).
An immunoglobulin light or heavy chain variable region (also referred to herein as a “light chain variable domain” (“VL domain”) or “heavy chain variable domain” (“VH domain”), respectively) consists of a “framework” region interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs.” The framework regions serve to align the CDRs for specific binding to an epitope of an antigen. Thus, the term “hypervariable region” or “CDR” refers to the amino acid residues of an antibody that are primarily responsible for antigen binding. From amino-terminus to carboxyl-terminus, both VL and VH domains comprise the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989. Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. CDRs 1, 2, and 3 of a VL domain are also referred to herein, respectively, as CDR-L1, CDR-L2, and CDR-L3; CDRs 1, 2, and 3 of a VH domain are also referred to herein, respectively, as CDR-H1, CDR-H2, and CDR-H3.
Unless the context dictates otherwise, the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
The term “chimeric antibody” refers to an antibody having variable domains derived from a first species and constant regions derived from a second species. Chimeric immunoglobulins or antibodies can be constructed, for example by genetic engineering, from immunoglobulin gene segments belonging to different species. The term “humanized antibody,” as defined infra, is not intended to encompass chimeric antibodies. Although humanized antibodies are chimeric in their construction (i.e., comprise regions from more than one species of protein), they include additional features (i.e., variable regions comprising donor CDR residues and acceptor framework residues) not found in chimeric immunoglobulins or antibodies, as defined herein.
The term “humanized VH domain” or “humanized VL domain” refers to an immunoglobulin VH or VL domain comprising some or all CDRs entirely or substantially from a non-human donor immunoglobulin (e.g., a mouse or rat) and variable region framework sequences entirely or substantially from human immunoglobulin sequences. The non-human immunoglobulin providing the CDRs is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor.” In some instances, humanized antibodies may retain non-human residues within the human variable domain framework regions to enhance proper binding characteristics (e.g., mutations in the frameworks may be required to preserve binding affinity when an antibody is humanized).
A “humanized antibody” is an antibody comprising one or both of a humanized VH domain and a humanized VL domain. Immunoglobulin constant region(s) need not be present, but if they are, they are entirely or substantially from human immunoglobulin constant regions.
Specific binding of an antibody to its target antigen means an affinity of at least 106, 107, 108, 109, or 1010 M−1. Specific binding is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit (e.g., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Specific binding does not, however, necessarily imply that a monoclonal antibody binds one and only one target.
With regard to proteins as described herein, reference to amino acid residues corresponding to those specified by SEQ ID NO includes post-translational modifications of such residues.
The term “diluent” as used herein refers to a solution suitable for altering or achieving an exemplary or appropriate concentration or concentrations as described herein.
The term “container” refers to something into which an object or liquid can be placed or contained, e.g., for storage (for example, a holder, receptacle, vessel, or the like).
The term “administration route” includes art-recognized administration routes for delivering a therapeutic protein such as, for example, parenterally, intravenously, intramuscularly, or subcutaneously. For administration of an antibody for the treatment of cancer, administration into the systemic circulation by intravenous or subcutaneous administration may be desired. For treatment of a cancer characterized by a solid tumor, administration can also be localized directly into the tumor, if so desired.
The term “treatment” refers to the administration of a therapeutic agent to a patient, who has a disease with the purpose to cure, heal, alleviate, delay, relieve, alter, remedy, ameliorate, improve or affect the disease.
The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
The term “effective amount,” “effective dose,” or “effective dosage” refers to an amount that is sufficient to achieve or at least partially achieve the desired effect, e.g., sufficient to inhibit the occurrence or ameliorate one or more symptoms of a disease or disorder. An effective amount of a pharmaceutical composition is administered in an “effective regime.” The term “effective regime” refers to a combination of amount of the composition being administered and dosage frequency adequate to accomplish prophylactic or therapeutic treatment of the disease or disorder.
As used herein, the term “about” denotes an approximate range of plus or minus 10% from a specified value. For instance, the language “about 20 μg/Kg” encompasses a range of 18-22 μg/Kg. As used herein, about also includes the exact amount. Hence “about 20 μg/Kg” means “about 20 μg/Kg” and also “20 μg/Kg.”
This disclosure provides description of the activity of a non-fucosylated anti-CD40 antibody, SEA-CD40. SEA-CD40 is an agonistic antibody and has enhanced binding to Fcγ receptors III and, surprisingly exhibits enhanced activation of the CD40 signaling pathway. Because of its enhanced activation of the CD40 pathway SEA-CD40 is a potent activator of the immune system and can be used to treat cancer or to treat infectious diseases, particularly chronic viral diseases, such as hepatitis C, human immunodeficiency virus, Epstein-Barr virus, cytomegalovirus, John Cunningham virus, and human papilloma virus. Other infectious diseases, include, e.g., tuberculosis. The enhanced activation of the immune system allows SEA-CD40 to be dosed at low levels, as compared to a fucosylated parent antibody.
CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily. It is a single chain type I transmembrane protein with an apparent MW of 50 kDa. Its mature polypeptide core consists of 237 amino acids, of which 173 amino acids comprise an extracellular domain (ECD) organized into 4 cysteine-rich repeats that are characteristic of TNF receptor family members. Two potential N-linked glycosylation sites are present in the membrane proximal region of the ECD, while potential O-linked glycosylation sites are absent. A 22 amino acid transmembrane domain connects the ECD with the 42 amino acid cytoplasmic tail of CD40. Sequence motifs involved in CD40-mediated signal transduction have been identified in the CD40 cytoplasmic tail. These motifs interact with cytoplasmic factors called TNF-R-associated factors (TRAFs) to trigger multiple downstream events including activation of MAP kinases and NFκB, which in turn modulate the transcriptional activities of a variety of inflammation-, survival-, and growth-related genes. See, e.g., van Kooten and Banchereau, J. Leukoc. Biol. 67:2-17 (2000); Elgueta et al., Immunol. Rev. 229:152-172 (2009).
Within the hematopoietic system, CD40 can be found on B cells at multiple stages of differentiation, monocytes, macrophages, platelets, follicular dendritic cells, dendritic cells (DC), eosinophils, and activated T cells. In normal non-hematopoietic tissues, CD40 has been detected on renal epithelial cells, keratinocytes, fibroblasts of synovial membrane and dermal origins, and activated endothelium. A soluble version of CD40 is released from CD40-expressing cells, possibly through differential splicing of the primary transcript or limited proteolysis by the metalloproteinase TNFα converting enzyme. Shed CD40 can potentially modify immune responses by interfering with the CD40/CD40L interaction. See, e.g., van Kooten and Banchereau, J. Leukoc. Biol. 67:2-17 (2000); Elgueta et al., Immunol. Rev. 229:152-172 (2009).
The endogenous ligand for CD40 (CD40L) is a type II membrane glycoprotein of 39 kDa also known as CD154. CD40L is a member of the TNF superfamily and is expressed as a trimer on the cell surface. CD40L is transiently expressed on activated CD4+, CD8+, and γδ T cells. CD40L is also detected at variable levels on purified monocytes, activated B cells, epithelial and vascular endothelial cells, smooth muscle cells, and DCs, but the functional relevance of CD40L expression on these cell types has not been clearly defined (van Kooten 2000; Elgueta 2009). However, expression of CD40L on activated platelets has been implicated in the pathogenesis of thrombotic diseases. See, e.g., Ferroni et al., Curr. Med. Chem. 14:2170-2180 (2007).
The best-characterized function of the CD40/CD40L interaction is its role in contact-dependent reciprocal interaction between antigen-presenting cells and T cells. See, e.g., van Kooten and Banchereau, J. Leukoc. Biol. 67:2-17 (2000); Elgueta et al., Immunol. Rev. 229:152-172 (2009). Binding of CD40L on activated T cells to CD40 on antigen-activated B cells not only drives rapid B cell expansion, but also provides an essential signal for B cells to differentiate into either memory B cells or plasma cells. CD40 signaling is responsible for the formation of germinal centers in which B cells undergo affinity maturation and isotype switching to acquire the ability to produce high affinity antibodies of the IgG, IgA, and IgE isotypes. See, e.g., Kehry, J. Immunol. 156:2345-2348 (1996). Thus, individuals with mutations in the CD40L locus that prevent functional CD40/CD40L interaction suffer from the primary immunodeficiency X-linked hyper-IgM syndrome that is characterized by over-representation of circulating IgM and the inability to produce IgG, IgA, and IgE. These patients demonstrate suppressed secondary humoral immune responses, increased susceptibility to recurrent pyrogenic infections, and a higher frequency of carcinomas and lymphomas. Gene knockout experiments in mice to inactivate either CD40 or CD40L locus reproduce the major defects seen in X-linked hyper-IgM patients. These KO mice also show impaired antigen-specific T cell priming, suggesting that the CD40L/CD40 interaction is also a critical factor for mounting cell-mediated immune responses. See, e.g., Elgueta et al., Immunol. Rev. 229:152-172 (2009).
The immune-stimulatory effects of CD40 ligation by CD40L or anti-CD40 in vivo have correlated with immune responses against syngeneic tumors. See, e.g., French et al., Nat. Med. 5:548-553 (1999). A deficient immune response against tumor cells may result from a combination of factors such as expression of immune checkpoint molecules, such as PD-1 or CTLA-4, decreased expression of MHC antigens, poor expression of tumor-associated antigens, appropriate adhesion, or co-stimulatory molecules, and the production of immunosuppressive proteins like TGFβ by the tumor cells. CD40 ligation on antigen presenting and transformed cells results in up-regulation of adhesion proteins (e.g., CD54), co-stimulatory molecules (e.g., CD86) and MHC antigens, as well as inflammatory cytokine secretion, thereby potentially inducing and/or enhancing the antitumor immune response, as well as the immunogenicity of the tumor cells. See, e.g., Gajewski et al., Nat. Immunol. 14:1014-1022 (2013).
A primary consequence of CD40 cross-linking is DC activation (often termed licensing) and potentiation of myeloid and B cells ability to process and present tumor-associated antigens to T cells. Besides having a direct ability to activate the innate immune response, a unique consequence of CD40 signaling is APC presentation of tumor-derived antigens to CD8+ cytotoxic T cell (CTL) precursors in a process known as ‘cross-priming’. This CD40-dependent activation and differentiation of CTL precursors by mature DCs into tumor-specific effectors CTLs may enhance cell-mediated immune responses against tumor cells. See, e.g., Kurts et al., Nat. Rev. Immunol. 10:403-414 (2010).
Agonistic CD40 mAbs including dacetuzumab, the SEA-CD40 parent molecule, have shown encouraging clinical activity in single-agent and combination chemotherapy settings. Dacetuzumab demonstrated some clinical activity in a phase 1 study in NHL and a phase 2 study in diffuse large B-cell lymphoma (DLBCL). See, e.g., Advani et al., J. Clin. Oncol. 27:4371-4377 (2009) and De Vos et al., J. Hematol. Oncol. 7:1-9 (2014). Additionally CP-870,893, a humanized IgG2 agonist antibody to CD40, showed encouraging activity in solid tumor indications when combined with paclitaxel or carboplatin or gemcitabine. In these studies, activation of antigen presenting cells, cytokine production, and generation of antigen-specific T cells were seen. See, e.g., Beatty et al., Clin. Cancer Res. 19:6286-6295 (2013) and Vonderheide et al., Oncoimmunology 2:e23033 (2013).
Because of its role in immune function, antibodies have been raised against the CD40 antigen. Such antibodies can be classified into three groups, antagonistic antibodies, which inhibit CD40 activity; partially agonistic antibodies, which partially induce CD40 activity; and fully agonistic antibodies, which fully stimulate CD40 activity. Members of each of the groups have been tested in clinical trials; none have been approved to date.
SEA-CD40
This disclosure provides a non-fucosylated hS2C6 antibody, SEA-CD40. S2C6 was originally isolated as a murine monoclonal antibody raised against a human bladder carcinoma referred to herein as mS2C6. See, e.g., Paulie et al., Cancer Immunol. Immunother. 17:165-179 (1984). The S2C6 antibody is a partial agonist of the CD40 signaling pathway and thus has the following activities: binding to human CD40 protein, binding to cynomolgus CD40 protein, activation of the CD40 signaling pathway, potentiation of the interaction of CD40 with its ligand, CD40L. See, e.g., U.S. Pat. No. 6,946,129.
As a next step in development, S2C6 was humanized and this humanized antibody is referred to as humanized S2C6, herein, and alternatively as dacetuzumab, or fucosylated, humanized S2C6 (fhS2C6), or SGN-40. See, e.g., WO 2006/128103. SGN-40 was tested in human clinical trials and was found not to be sufficiently active to warrant further development.
SEA-CD40 is a non-fucosylated humanized S2C6 antibody. The amino acid sequences of the heavy and light chain for SEA-CD40 are disclosed as SEQ ID NO:1 and 2, respectively. The variable region of the heavy chain is from amino acids 1-113 of SEQ ID NO:1; the variable region of the light chain is from amino acids 1-113 of SEQ ID NO:2. The generation of the antibody backbone of SEA-CD40 is disclosed at WO 2006/128103, which is herein incorporated by reference.
This disclosure provides a non-fucosylated, humanized S2C6 antibody, referred to herein as of hS2C6 or SEA-CD40. In addition to enhanced binding to Fc receptors, SEA-CD40 also enhances activity of the CD40 pathway, as compared to the parent antibody, dacetuzumab. The SEA-CD40 antibody thus, is administered to patients at lower doses and using different schedules of administration.
SEA-CD40 is a non-fucosylated antibody and exhibits enhanced binding to FcγIII receptors, and surprisingly enhanced ability to activate the CD40 signaling pathway in immune cells.
Methods of Making Non Fucosylated Antibodies
This disclosure provides compositions and methods for preparing humanized S2C6 antibodies with reduced core fucosylation. As used herein, “core fucosylation” refers to addition of fucose (“fucosylation”) to N-acetylglucosamine (“GlcNAc”) at the reducing terminal of an N-linked glycan.
Fucosylation of complex N-glycoside-linked sugar chains bound to the Fc region (or domain) of the SEA-CD40 antibody backbone is reduced. As used herein, a “complex N-glycoside-linked sugar chain” is typically bound to asparagine 297 (according to the EU index as set forth in Kabat, “Sequences of Immunological Interest, 5th Ed., Pub. No. 91-3242, U.S. Dept. Health & Human Services, NIH, Bethesda, Md., 1991). As used herein, the complex N-glycoside-linked sugar chain has a biantennary composite sugar chain, mainly having the following structure:
where ± indicates the sugar molecule can be present or absent, and the numbers indicate the position of linkages between the sugar molecules. In the above structure, the sugar chain terminal which binds to asparagine is called a reducing terminal (at right), and the opposite side is called a non-reducing terminal. Fucose is usually bound to N-acetylglucosamine (“GlcNAc”) of the reducing terminal, typically by an α1,6 bond (the 6-position of GlcNAc is linked to the 1-position of fucose). “Gal” refers to galactose, and “Man” refers to mannose.
A “complex N-glycoside-linked sugar chain” includes 1) a complex type, in which the non-reducing terminal side of the core structure has one or more branches of galactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAc optionally has a sialic acid, bisecting N-acetylglucosamine or the like; or 2) a hybrid type, in which the non-reducing terminal side of the core structure has both branches of a high mannose N-glycoside-linked sugar chain and complex N-glycoside-linked sugar chain.
In some embodiments, the “complex N-glycoside-linked sugar chain” includes a complex type in which the non-reducing terminal side of the core structure has zero, one or more branches of galactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAc optionally further has a structure such as a sialic acid, bisecting N-acetylglucosamine or the like.
According to the present methods, typically only a minor amount of fucose is incorporated into the complex N-glycoside-linked sugar chain(s) of the SEA-CD40 molecule. For example, in various embodiments, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 3% of the antibody has core fucosylation by fucose. In some embodiments, about 2% of the antibody has core fucosylation by fucose.
In certain embodiments, only a minor amount of a fucose analog (or a metabolite or product of the fucose analog) is incorporated into the complex N-glycoside-linked sugar chain(s). For example, in various embodiments, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 3% of the SEA-CD40 antibody has core fucosylation by a fucose analog or a metabolite or product of the fucose analog. In some embodiments, about 2% of the SEA-CD40 antibody has core fucosylation by a fucose analog or a metabolite or product of the fucose analog.
Methods of making non-fucosylated antibodies by incubating antibody-producing cells with a fucose analogue are described, e.g., in WO/2009/135181. Briefly, cells that have been engineered to express the humanized S2C6 antibody are incubated in the presence of a fucose analogue or an intracellular metabolite or product of the fucose analog. As used herein, an intracellular metabolite can be, for example, a GDP-modified analog or a fully or partially de-esterified analog. A product can be, for example, a fully or partially de-esterified analog. In some embodiments, a fucose analogue can inhibit an enzyme(s) in the fucose salvage pathway. For example, a fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit the activity of fucokinase, or GDP-fucose-pyrophosphorylase. In some embodiments, a fucose analog (or an intracellular metabolite or product of the fucose analog) inhibits fucosyltransferase (preferably a 1,6-fucosyltransferase, e.g., the FUT8 protein). In some embodiments, a fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit the activity of an enzyme in the de novo synthetic pathway for fucose. For example, a fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit the activity of GDP-mannose 4,6-dehydratase or/or GDP-fucose synthetase. In some embodiments, the fucose analog (or an intracellular metabolite or product of the fucose analog) can inhibit a fucose transporter (e.g., GDP-fucose transporter).
In one embodiment, the fucose analogue is 2-flurofucose. Methods of using fucose analogues in growth medium and other fucose analogues are disclosed, e.g., in WO/2009/135181, which is herein incorporated by reference.
Other methods for engineering cell lines to reduce core fucosylation included gene knock-outs, gene knock-ins and RNA interference (RNAi). In gene knock-outs, the gene encoding FUT8 (alpha 1,6-fucosyltransferase enzyme) is inactivated. FUT8 catalyzes the transfer of a fucosyl residue from GDP-fucose to position 6 of Asn-linked (N-linked) GlcNac of an N-glycan. FUT8 is reported to be the only enzyme responsible for adding fucose to the N-linked biantennary carbohydrate at Asn297. Gene knock-ins add genes encoding enzymes such as GNTIII or a golgi alpha mannosidase II. An increase in the levels of such enzymes in cells diverts monoclonal antibodies from the fucosylation pathway (leading to decreased core fucosylation), and having increased amount of bisecting N-acetylglucosamines. RNAi typically also targets FUT8 gene expression, leading to decreased mRNA transcript levels or knocking out gene expression entirely. Any of these methods can be used to generate a cell line that would be able to produce a non-fucosylated antibody, e.g., an SEA-CD40 antibody.
Those of skill will recognize that many methods are available to determine the amount of fucosylation on an antibody. Methods include, e.g., LC-MS via PLRP-S chromatography and electrospray ionization quadrupole TOF MS.
The non-fucosylated antibody, SEA-CD40, when adminstered to a patient induces activation of monocyte maturation into macrophages and induce production of cytokines, including, e.g., interferon-γ (IFN-γ) and chemokine that elicit robust T-cell response to immune system challenges. Unlike fully agoninstic antibodies, such as antibody 24.4.1, SEA-CD40 does not induce production of immune-dampening cytokines, such as interleukin-10 (IL-10). IL-10, in turn, induces activity of T-regulatory cells, which dampen the immune response. Thus, SEA-CD40 is useful for induction of a robust T-cell mediated immune response without promoting activity of T-regulatory cells.
Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection. The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
SEA-CD40 is administered intravenously. In other embodiments, SEA-CD40 is administered subcutaneously. In a further embodiment, SEA-CD40 is administered subcutaneously at the site of a tumor.
The non-fucosylated SEA-CD40 antibody has surprisingly enhanced immune activation activity as compared to its parent antibody, dacetuzumab. Thus, SEA-CD40 can be administered to patients at lower doses and on different schedules as compared to dacetuzumab.
As an example, SEA-CD40 can be adminstered to patients at levels between 0.1-2000 μg/kg (μg antibody per kilogram patient body weight). Other possible dosage ranges are 10-1000 μg/kg, 50-800 μg/kg, 75-600 μg/kg, 100-500 μg/kg. Other possible dosage ranges are the following: 100-300 μg/kg, 300-500 μg/kg, 500-700 μg/kg, 700-900 μg/kg, and 900-1100 μg/kg. Still more dose ranges are the following: 100-150 μg/kg, 150-200 μg/kg, 200-250 μg/kg, 250-300 μg/kg, 300-350 μg/kg, 350-400 μg/kg, 400-450 μg/kg, 450-500 μg/kg, 500-550 μg/kg, 550-600 μg/kg, 600-650 μg/kg, 650-700 μg/kg, 700-750 μg/kg, 750-800 μg/kg, 800-850 μg/kg, 850-900 μg/kg, 900-950 μg/kg, 950-1000 μg/kg, 1000-1050 μg/kg, and 1050-1100 μg/kg. Other possible dosage ranges are 0.3-200 μg/kg, 0.6-150 μg/kg, 1.0-100 μg/kg, 2-50 μg/kg, 5-25 μg/kg, 7.5-15 μg/kg, and 8-12 μg/kg.
In other embodiments, SEA-CD40 is administered to patients at 0.6 μg/kg, 1.0 μg/kg, 2.5 μg/kg, 5.0 μg/kg, 7.5 μg/kg, 10 μg/kg, 30 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg, or 200 μg/kg. In a preferred embodiment, SEA-CD40 is administered to patients at 10 μg/kg.
In further embodiments, SEA-CD40 is administered to patients at about 60 μg/kg, about 100 μg/kg, about 150 μg/kg, about 200 μg/kg, about 250 μg/kg, about 300 μg/kg, about 350 μg/kg, about 400 μg/kg, about 450 μg/kg, about 500 μg/kg, about 550 μg/kg, about 600 μg/kg, about 650 μg/kg, about 700 μg/kg, about 750 μg/kg, about 800 μg/kg, about 850 μg/kg, about 900 μg/kg, about 950 μg/kg, about 1000-1050 μg/kg, about 1050 μg/kg, and 1110 μg/kg.
In some embodiments, SEA-CD40 is administered in a manner to reduce the likelihood of immune exhaustion. For example, SEA-CD40 can be administered at three week intervals, six week intervals, eight week intervals, ten week intervals, twelve week intervals, or 14 wek intervals. Intervals can also be on a monthly schedule, e.g., one month intervals, two month intervals, or three month intervals.
Because SEA-CD40 activates the immune system to respond against tumor-related antigens, its use is not limited to cancers that express CD40. Thus SEA-CD40 can be used to treat both CD40 positive and CD40 negative cancers.
SEA-CD40 is preferably used to treat tumors that are known to be immune responsive, particularly if the cancer expresses low levels of CD40 or does not detectably express CD40. Immune responsive cancers include, e.g., melanoma; bladder cancer; lung cancer, e.g., small cell lung cancer and non-small cell lung cancer; ovarian cancer; kidney cancer; pancreatic cancer; breast cancer; cervical cancer; head and neck cancer, prostate cancer; glioblastoma; non-hodgkin lymphoma; chronic lymphocytic leukemia; hepatocellular carcinoma; or multiple myeloma.
In another embodiment, SEA-CD40 is used to treat solid tumors. In a further embodiment, SEA-CD40 is used to treat blood cancers, e.g., lymphoma, including non-Hodgkin lymphoma and Hodgkin lymphoma; chronic lymphocytic leukemia; or multiple myeloma.
Because of its immune stimulatory function, SEA-CD40 can be used in combination with other therapeutic agents that activate the immune system. Drugs with immune stimulatory function include, e.g., T-cell modulators, including immune checkpoint inhibitors; immune activators; and chemotherapeutic agents that induce immunogenic cell death. As an example, certain antibodies function by blocking activity of molecules that serve as immune checkpoints on T cells. SEA-CD40 can, therefore be used in combination with antibodies that target immune checkpoint proteins.
T-Cell Modulators
T-cells play a role in the ability of the immune system to recognize and eliminate cancers from the body. T-cell modulators include antibodies that block the function of immune checkpoints. See, e.g., Pardoll, Nature Rev. Cancer, 12:252-264 (2012). Antibodies that block immune checkpoints include, e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA4 antibodies. Other checkpoint inhibitors/activators include LAG3 and TIM3. Antibodies against some proteins can be used to modulate T-cell activity or preferably activate T-cell activity, e.g., antibodies against 41BB, CD27, ICOS, and OX40. Other T-cell modulators include inhibitors of the enzyme indolamine 2,3-dioxygenase (DO).
Anti-CTLA4 antibodies recognize the protein cytotoxic lymphocyte 4 (CTLA-4), also known as cluster of differentiation 152 or CD152. The CTLA-4 protein is expressed on T cells, which recognize antigens that are suitable for attack by the immune system. Activation of CTLA-4 dampens the immune response. See e.g., Nirschi and Drake, Clin. Cancer Res., 19:4917-4924 (2013). Antibodies specific for CTLA-4 and that block its activity have been used to treat cancer by upregulating the immune response to cancers. Examples of CTLA-4 antibodies include ipilimumab or tremelimumab. SEA-CD40 can be administered in combination with ipilimumab or tremelimumab to treat cancer.
Anti-PD1 antibodies recognize the protein programmed death-1 (PD-1). Like CTLA-4, PD-1 is expressed on T cells, and dampens the immune response. See e.g., Nirschi and Drake, Clin. Cancer Res., 19:4917-4924 (2013). Antibodies specific for PD-1 and that block its activity have been used to treat cancer by upregulating the immune response to cancers. Examples of PD-1 antibodies include MEDI0680, AMP-224, nivolumab, pembrolizumab, and pidilizumab. Other PD-1 binding proteins that act as checkpoint inhibitors and can be used in combination with SEA-CD40 include, e.g., B7-DC-Fc. SEA-CD40 can be administered in combination with MEDI0680, AMP-224, nivolumab, pembrolizumab, or pidilizumab to treat cancer.
PD-L1 is a ligand of the PD-1 protein. PD-L1 is expressed on cancer cells and its interaction with PD-1 allows PD-L1-expressing cancer cells to evade the immune system. Anti-PD-L1 antibodies have been generated and used to treat cancer. Examples of PD-L1 antibodies include, e.g., MEDI4736, BMS-936559/MDX-1105, MSB0010718C and MPDL3280A. SEA-CD40 can be administered in combination with MEDI4736, BMS-936559/MDX-1105, MSB0010718C or MPDL3280A to treat cancer.
Other antibodies that block the function of immune checkpoint proteins include antibodies directed against e.g., LAG3 and TIM13, and can be used in combination with SEA-CD40.
Antibodies against 41BB, CD27, ICOS, and OX40 are used to activate T-cell activity and can be used in combination with SEA-CD40. OX40 antibodies include, e.g., MEDI6469 and MEDI6383. An example of an agonistic anti-CD27 antibody is CDX-1127, which can be used in combination with SEA-CD40.
The enzyme indolamine 2,3-dioxygenase (DO) catalyzes the degradation of the amino acid tryptophan. Inhibitors of DO can be small molecules, such as rosmarinic acid, COX-2 inhibitors, and alpha-methyl-tryptophan.
Chemotherapeutic Agents that Induce Immunogenic Cell Death
In most humans, millions of cells die via apoptosis and are removed without generating an immune response. However, after treatment with some chemotherapeutic agents, immune cells have been observed to infiltrate tumors. Thus, some tumor cells killed by chemotherapeutic agents act as vaccines and raise a tumor-specific immune response. This phenomenon is referred to as immunogenic cell death (ICD). See, e.g., Kroemer et al., Annu. Rev. Immunol., 31:51-72 (2013). The ability of a chemotherapeutic agent to induce ICD can be determined experimentally. Two criteria must be met. First, injection of an immunocompetent mouse with cancer cells that have been treated in vitro with a chemotherapeutic agent must elicit a protective immune response that is specific for tumor antigens, in the absence of adjuvant. Second, ICD occurring in vivo, e.g., a mouse syngeneic model with treatment using a potential ICD-inducing chemotherapeutic agent, must drive an immune response in the tumor that is dependent on the immune system.
Chemotherapeutic agents that induce ICD include, e.g., anthracyclines, anti-EGFR antibodies, bortezomib, cyclophosphamide, gemcitabine, irradiation of the tumor, and oxaliplatin. SEA-CD40 can be used in combination with any of these agents to generate an enhanced immune response and treat cancer in a patient.
Immune Activation
Cancer can is also treated by administering agents that directly stimulate the immune system. Such agents include, e.g., GM-CSF, IFN-gamma, interleukin-2, GVAX, and TLR9 agonists. Other immune activators include, e.g., cancer vaccines, Bacillus Calmette-Guérin (BCG), nonspecific immunostimulants (e.g. imiquimod) and cellular therapies like CAR-T cells. SEA-CD40 can be used in combination with any of these agents to generate an enhanced immune response and treat cancer in a patient.
Other Combinations
Other combinations with SEA-CD40 can be used to treat cancer. Examples include, e.g., SEA-CD 40 in combination with an anti-PD1 antibody, e.g., nivolumab, pembrolizumab, and pidilizumab, MEDI0680, or AMP-224; SEA-CD40 in combination with Gemcitabine, with or without paclitaxel or cisplatin or oxaliplatin; SEA-CD-40 in combination with a BRAF inhibitor, e.g., vemurafenib or dabrafenib; or SEA-CD40 in combination with cyclophosphamide, ADRIAMYCIN™, vincristine, and prednisone (CHOP) or rituximab, ifosfamide, carboplatin, and etopiside (RICE) or rituximab, gemcitabine, dexamethasone and cisplatin (RGDP).
The following examples are offered to illustrate, but not to limit the claimed invention.
The humanized anti-CD40 antibody, S2C6 with heavy and light light chains of SEQ ID NOs: 1 and 2 was expressed in CHO cells. A fucosylation inhibitor, 2-fluorofucose, was included in the cell culture media during the production of antibodies resulted in non-fucosylated antibody, SEA-CD40. See, e.g., Okeley et al., Proc. Nat'l Acad. Sci. 110:5404-55409 (2013). The base media for cell growth was fucose free and 2-flurofucose was added to the media to inhibit protein fucosylation. Ibid. Incorporation of fucose into antibodies was measured by LC-MS via PLRP-S chromatography and electrospray ionization quadrople TOF MS. Ibid. Data not shown.
CD40 Binding affinity determination of SEA-CD40: For isolation of peripheral blood mononuclear cells (PBMCs), human whole blood was supplied by ASTARTE BIOLOGICS™. Briefly, blood was collected into heparin tubes and delivered to SEATTLE GENETICS™ within four hours of being drawn. Upon arrival blood was aliquoted into 50 ml conical tubes (falcon) and spun at 200 g in an EPPENDORF™ 5810R (A-4-62 rotor) for 20 minutes at 25° C., without break to separate the platelet rich fraction. Following centrifugation, three distinct layers were formed: bottom layer, red blood cells (accounting for 50-80% of the total volume); middle layer, very thin band of white blood cells; top layer, straw-colored platelet rich plasma (PRP).
The upper straw colored layer with which is enriched in platelets was removed with a one ml pipette. Once the platelet rich plasma was removed blood was diluted with equal volumes of sterile PBS (GIBCO™, lot 1618435, ept 2016 July). 15 mls of HISTOPAQUE™-1077 (SIGMA™, lot number RNBD2965, Expt. May 2017) warmed to room temperature was underlayered below the blood. HISTOPAQUE™ samples were spun at 1500 rpm for 25 minutes at 25° C. with outbreak. Following centrifugation three layers are formed again: bottom layer, red blood cells (accounting for 50-80% of the total volume); middle layer, thick band of white blood cells (also called “buffy coat”); top layer, PBS and remaining platelets.
The upper PBS/Platelet layer was removed with a 1 ml pipet and discarded. The thick band of white blood cells was gently removed and placed into a clean 50 ml sterile conical tube. Tubes were filled to 50 mls and cells are spun at 800 g for 10 minutes. Wash solution was removed and pellets were resuspended in 10 mls of ACK red blood lysis buffer (GIBCO™, lot 1618488) for ten minutes. Fifty milliliter conical tubes were then topped off with 35 ml sterile PBS and cells were spun at 800 g for ten minutes. The wash solution was removed and pellet was resuspended in 50 mls of PBS. Five hundred μl of sample was removed and PBMC were counted with a Vi-cell-XR (BECKMAN COULTER™). Cells were spun again at 800 g for ten minutes. The wash solution was removed and pellet re-suspended at 1×106/ml in FACs staining solution (BD). One hundred μl of resuspended PBMC's were plated into a 96 well U-bottom plate (CORNING™) and placed on ice. To block non-specific FcγRIIIa binding, PBMC's were pre-treated 100 μg/ml of human Fc-fragments (CALBIOCHEM™) for thirty minutes. Ten-fold serial dilutions of biotinylated SEA-h00 (non-fucosylated control antibody), SEA-CD40, or SGN-40 were prepared to create a dilution series of (100, 10, 1, 0.1, 0.01, 0.001, 0.0001 μg/ml).
Samples were washed twice in ice cold FACs buffer and incubated with saturating concentrations of PE-Streptavidin (BD™) on ice for thirty minutes. Samples were washed twice in ice cold FACs buffer and re-suspended in 200 μl of FAC's buffer. Binding was assessed using a BD LSRII and DIVA software. FCS were analyzed in FLOWJO™ and GeoMean fluorescence of positively stained cells was determined and plotted in Prism Graph Pad. Data was fit to nonlinear regression assuming one binding site in Prism and binding KD values calculated by dividing μg/ml calculation by molecular weight of SEA-CD40.
Results: The binding affinity of SEA-CD40, and the parental antibody dacetuzumab, to CD40 on human peripheral blood mononuclear cells (PBMC) was determined by flow cytometry. Background binding of an appropriate isotype control was subtracted and mean fluorescence intensity (MFI) was plotted against antibody concentration. Results are shown in
FcγRIIIa Binding affinity determination of SEA-CD40: CHO cells that express the high (158V) or low (158F) version of human FcγRIIIa were generated. 20×106 cells were centrifuged, washed once in 20 ml 1×PBS, and resuspended in 8 ml BD stain buffer. Cells were aliquoted in the following density: 2.0×106 cells/ml in 100 μl volume. 0.20×106 cells were aliquoted to each well. Cells were centrifuged at 1250 rpm, for five minutes at room temperature.
Antibodies were diluted to either 0.14 ug/ml (SGN) or 0.04 ug/ml (SEA). Dilutions are provided in Table 1.
Supernatants were aspirated from the spun cells and 60 μl of corresponding antibody dilutions were added with a multichannel pipet. Corresponding concentrations were 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.14 mcg/ml. Samples were incubated at 4° C. for 1 hour. Samples were centrifuged, and washed twice with 200 μl BD stain buffer per well. One milliliter of Streptavidin-PE was added to 20 ml BD™ stain buffer (excess 2°) to make streptavidin buffer. 100 μl of streptavidin buffer was added to each sample and they were incubated for 30 min in the dark at 4° C. Samples were then centrifuged and washed twice with 200 ul BD™ Stain buffer per well. Samples were analyzed by Flow cytometry in HTS mode on the LSRII and graph MFI to calculate Kd's in PRIZM.
Results: Binding of SEA-CD40 and the parent antibody dacetuzumab to Chinese hamster ovary (CHO) cells expressing the low (158F) or high (158V) affinity form of FcγRIIIa was assessed. Results are shown in
SEA-CD40 mediated ADCC activity: Human PBMC's were isolated as above and were treated with various concentrations of SEA-CD40 or an SEA-isotype control (SEA-h00) for 6, 24, or 48 hours. Cultures were stained for CD19+ B cells and cell numbers were quantified by flow cytometry.
Results: Human PBMC cultures, were treated with 100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 μg/mL of SEA CD40 or a non-binding SEA-isotype control (SEA-h00) for 6, 24, and 48 hours and the number of CD40 positive cells were then assessed. Results are shown in
Assessment of cytokine production in human whole blood or PBMCs: Human whole blood was supplied by ASTARTE BIOLOGICS™. Briefly, 100 mls of blood was collected into heparin tubes and delivered to SEATTLE GENETICS™ within 4 hours of the draw. Half of the blood was set aside for whole blood cultures while the other half was used to isolate PBMCs as described above. One hundred μl of whole blood was aliquoted into 3-96 flat bottom tissue culture plates (COSTAR™). Isolated PBMCs were counted in a VIACELL™ and resuspended at 1×106 cells/ml in DMEM containing 10% FBS (ASTARTE BIOLOGICS™), 1× penicillin/strepA, and X glutamine (PBMC media). For PBMCs, one hundred μl of resuspened, purified PBMCs were aliquoted into 3-96 flat bottom tissue culture plates. 10× serial dilutions of SEA-h00 and SEA-CD40 were made in PBMC media and whole blood and isolated PBMC cultures were treated with descending concentrations of either SEA-h00 or SEA-CD40 (100, 10, 1.0, 0.1, 0.01, 0.001, 0.0001 or 0 μg/ml). SEA-CD40 treatment was performed in duplicate for both whole blood and PBMC cultures at each time point. At each of the pre-determined times points (6, 24, and 48 hours) a 96 well plate containing whole blood or purified PBMCs was spun with a plate adapter in an EPPENDORF™ 5810R at 800 rpm for 5 minutes. Serum or tissue culture supernatants were removed and transferred to a 96 strip tube rack and samples were frozen at −80° C. until processing.
Frozen tissue culture supernatants and serum were thawed overnight at 4° C. and processed for cytokine production using a LUMINEX™ multiplex Kit from MILLIPORE™ Custom kits were designed to analyze IFNγ, IL-12p40, IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, TNF-α, sCD40L. Analytes were picked based on cytokines observed with dacetuzemab in previous studies. Tissue culture supernatants and serum samples were processed as per the manufactures instructions. Briefly, assay plates were washed with 200 μL of wash buffer per well, followed by addition of 25 μL standard or buffer, 25 μL matrix or sample, and 25 μL of multiplexed analyte beads to each well. Samples were incubated overnight with vigorous shaking at 4° C. Plates were washed twice with wash buffer. Twenty-five μL of detection antibodies were added to each well and incubated at room temperature for one hour. Twenty-five μL of streptavidin-phycoerythrin (SA-PE) were added and samples incubated at room temperature for thirty minutes. The plate was washed twice with wash buffer and beads were resuspended with 150 μL of sheath fluid. The samples were analyzed using LUMINEX™ MAGPIX™ systems in combination with the XPONENT™ software system. Cytokine levels were calculated from the standard curve.
Results: Human whole blood cultures were treated with a SEA-isotype control or SEA-CD40 (100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 m/mL) for 6, 24, or 48 hours. Serum or tissue culture supernatants were collected and inflammatory cytokines assessed by multiplexed LUMINEX™ analysis. The data are plotted as a fold increase in cytokine production compared to a SEA-isotype control. SEA-CD40 stimulated robust production of IFNγ, MIP1β, and TNFα at 6, 24, and 48 hours in whole blood, as shown in Table 2, below. SEA-CD40 levels are provided in the leftmost columns. Activity was observed at levels as low as 0.010 m/mL SEA-CD40. Stimulation of MIP1β, and TNFα at twenty-four hours are shown in
Human PBMC were treated with a SEA-isotype control or SEA-CD40 (100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 μg/mL) for 6, 24, or 48 hours. Serum or tissue culture supernatants were collected and inflammatory cytokines assessed by multiplexed LUMINEX™ analysis. The data are plotted as a fold increase in cytokine production compared to a SEA-isotype control. SEA-CD40 stimulated robust production of IFNγ, MIP1β, and TNFα at 6, 24, and 48 hours PBMCs, as shown in Table 3, below. SEA-CD40 levels, in μg/mL, are provided in the leftmost columns. Activity was observed at levels as low as 0.010 μg/mL SEA-CD40. Stimulation of MIP1β, and TNFα at twenty-four hours are shown in
48 hrs
Assessment of Activation markers on PBMCs: Co-stimulatory molecule surface expression was assessed on the cell pellets remaining from the cytokine analysis described above. Cell pellets were resuspended in 50 ml of BD FACs buffer and transferred to and 96 well round bottomed microtiter plates Fc receptors were blocked with human 100 μg/ml Fc-fragments (MILLIPORE™) for 30 minutes on ice. A master mix composed of PE-CD86 (BD) and MHCII (Pan anti-DR,DP,DQ antibody BD) diluted at 1:100 was prepared in BD FACs buffer containing 100 mg/ml human Fc fragments. Five μl of the master mix was added to each well containing ninety μl and samples were incubated for one hour on ice. Cells were then spun at 400 g in a pre-cooled EPPENDORF™ 5810R centrifuge for five minutes. Supernatants were removed and cells washed with 200 ml of BD FACs buffer. Cells were washed twice and then resuspended in 200 ml of FACs buffer. Samples were then analyzed on an LSRII (BD™ biosciences) with DIVA software (BD™ biosciences). CD86 and MHCII geo mean fluorescence was assessed using FLOWJO™ analysis software. A ratio between SEA-h00 and SEA-CD40 was calculated and the fold change used to calculate a range of SEA-CD40 potency.
Results: Activation of CD40, in addition to eliciting cytokine production, promotes the maturation of antigen presenting cells. DC maturation can be followed by upregulation of activation markers including CD86 and MHCII. Human PBMC cultures were stimulated with SEA CD40 and an SEA-isotype control for 6, 24, or 48 hours and surface expression of MHC Class II antigens (HLA DR, DP, DQ) and CD86 was assessed. SEA-CD40 stimulation, but not the isotype control resulted in a significant increase in both MHCII (Table 4) and CD86 (data not shown) at concentrations as low as 0.01 μg/mL.
Role of fucose in immune activation by SEA-CD40: Human PBMCs were isolated as described above. PBMCs were treated with various concentrations of SEA-CD40, the parental antibody SGN40, or a F(ab)′2 version of SEA-CD40 and incubated for 24 hrs. For assessment of B cell depletion, BPMC cultures were stained with a PE-CD19 and B-cell numbers were quantified by flow cytometry. For assessment of cytokine production, PBMC tissue culture supernatants were collected and cytokine production assessed by multiplex analysis on the Luminex platform. IFNγ production is shown is shown in
Results: To assess B cell depletion, human PBMC cultures were treated with multiple concentrations of SEA-CD40, dacetuzumab, or SEA-CD40 F(ab′)2 for twenty-four hours. Results are shown in
Additionally immune activation endpoints (cytokines and APC activation/maturation markers) were assessed in SEA-CD40, dacetuzumab, and SEA-CD40 F(ab′)2 PBMC cultures stimulated for twenty-four hours. SEA-CD40 stimulation of both cytokine production (
Identification of active doses of SEA-CD40: SEA-CD40 is proposed to be active at dose levels that activate antigen-presenting cells, which can be characterized by upregulation of activation markers, such as MHC class I or II, or CD86. Activation markers on PBMCs following treatment with various concentrations of SEA-CD40 from 6 to 48 hours were assessed as described above (Assessment of Activation markers on PBMCs). The difference between treatments with isotype control SEA-h00 and SEA-CD40 for each activation marker were calculated and plotted versus SEA-CD40 concentrations and treatment time. The steepest response-concentration curves were observed at 24 hours for CD86 and MHCII and 48 hours for MHCI. Response-concentration data (24 hr CD86, 24 hr MCHII and 48 hr MHCI) were fitted by nonlinear regression using the following equation, where 0% and 100% responses are defined as the smallest and largest values in the data set for each activation marker. EC50 is the concentration of SEA-CD40 that gives a 50% response.
Results: Normalized response versus—log (concentration) and nonlinear fitted regression lines for activation markers are illustrated in
T-cell response generated by SEA-CD40: An anti-M1 T cell line was generated at ASTARTE BIOLOGICS™ from a HLA-A2 donor that was shown to be highly reactive to the M1 flu peptide. These cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and combined with autologous PBMCs. Cultures were stimulated with 10 μg/ml M1 flu peptide in the presence or absence of decreasing concentrations (1, 0.1, 0.01 μg/ml) of SEA-CD40 or dacetuzumab for 5 days. Cultures supernatants were collected and analyzed for cytokines by multiplex analysis on the LUMINEX™ platform and antigen specific T-cells were identified by M1 specific Tetramer binding.
Results: PBMC's from an HLA-A2 donor shown to be highly reactive to the M1 flu peptide were stimulated with M1 flu peptide in the presence or absence of decreasing concentrations of SEA-CD40 or dacetuzumab for five days. Results are shown in
T-cell response generated by SEA-CD40 in combination with anti-immune checkpoint inhibitor antibodies: An anti-M1 T cell line was generated at ASTARTE BIOLOGICS™ from a HLA-A2 donor that was shown to be highly reactive to the M1 flu peptide. These cells were labeled with CFSE and combined with autologous PBMCs. Cultures were stimulated with 10 μg/ml M1 flu peptide in the presence or absence of decreasing concentrations (1, 0.1, 0.01 μg/ml) of SEA-CD40 and/or 1 μg/ml of an anti-CTLA4 or an anti-PD-1 antibody for five days. Culture supernatants were collected and analyzed for cytokines by multiplex analysis on the LUMINEX™ platform and antigen specific T-cells were identified by M1 specific Tetramer binding.
Results: PBMC's from an HLA-A2 donor shown to be highly reactive to the M1 flu peptide were stimulated with M1 flu peptide in the presence or absence of decreasing concentrations of SEA-CD40 and/or a constant concentration of an anti-CTLA4 blocking antibody or an anti-PD-1 antibody. Results are shown in
T-cell response generated by SEA-CD40 in PCMCs from cancer patients: For assessment of anti-CD40 antibodies alone, PBMCs were isolated from 10 mls of tumor patient blood and 0.25 million cells were plated in a 24 well plate. Samples were treated with increasing concentrations of SEA-CD40 only, or 1 ug/ml of a combined peptide pool containing MageA1/MageA3/NY-ESO and increasing concentration of either SEA-CD40 or SGN-40. Samples were cultured in 10% CO2 at 37° C. for five days, tissue culture supernatants were collected and INF-γ levels were assessed.
For assessment of SEA-CD40 in combination with immune checkpoint blocking antibodies, PBMCs were isolated from 10 mls of blood from patients diagnosed with either breast, pancreatic, of melanoma cancer, and 0.25 million cells were plated in a 24 well plate. Samples were treated with increasing concentrations of SEA-CD40, 1 μg/ml of a combined peptide pool containing MageA1/MageA3/NY-ESO, and either 1 μg/ml of anti-PD1 or anti-CTLA4. Samples were cultured in 10% CO2 at 37° C. for 5 days, tissue culture supernatants were collected and INF-γ levels were assessed.
Results: PBMC's from donors were isolated from whole blood as described above. The donors were three patients diagnosed with melanoma, three patients diagnosed with breast cancer, and three patients diagnosed with pancreatic cancer. Donor PBMC's were stimulated with a pool of peptides of the common tumor antigen proteins (MAGEA1/MAGE3/NY-ESO) in the presence or absence of increasing concentrations of SEA-CD40 or SGN-40 for 5 days. Tissue culture supernatants were collected and INF-γ production was assessed. Results are shown in
PBMC's from donors were isolated from whole blood as described above. As above, the donors were three patients diagnosed with melanoma, three patients diagnosed with breast cancer, and three patients diagnosed with pancreatic cancer. Donor PBMC's were stimulated with a pool of peptides of the common tumor antigen proteins (MAGEA1/MAGE3/NY-ESO) in the presence or absence of increasing concentrations of SEA-CD40 and/or a constant concentration of an anti-CTLA4 or anti-PD1 blocking antibody. Results are shown in
Mouse models have been proven to be very useful in assessing efficacy and mechanisms of new cancer therapeutics. Study of SEA-CD40 in mouse models of cancer has been difficult because SEA-CD40 does not recognize murine CD40. Therefore, to assess the activity of the non-fucosylated anti-CD40 antibodies a syngeneic murine tumor model was developed. The murine functional equivalents of human IgG1 and human FcγRIII/CD16 are murine IgG2a and FcγRIV, respectively, and binding of murine IgG2a to murine FcγRIV mediates ADCC. See, e.g., Bruhns, Blood 119:5640-5649 (2012) and Nimmeriahn et al., Immunity 23:41-51 (2005). The rat antibody 1C 10 was used to generate a surrogate of SEA-CD40. See, e.g., Heath et al., Eur. J. Immunol. 24:1828-1834 (1994). Briefly, the VL and VH gene fragments of a rat monoclonal antibody that recognizes murine CD40, the 1C10 antibody were cloned in-frame 5′ to murine Ckappa and murine IgG2a CH1-CH2-CH3 fragments, respectively. Expression of the resulting genes in CHO cells generated a chimeric 1C10 antibody with rat VL and VH domains and murine light and heavy chain domains of the IgG2a isotype (mIgG2a 1C10). mIgG2a 1C10 was expressed in the presence of 2-fluorofucose in the CHO cell growth medium using the methods described in Example 1, to generate a non-fucosylated form of mIgG2a 1C10 (mIgG2a SEA 1C10). Fucosylated mIgG2a 1C10 and mIgG2a SEA 1C10 were tested for anti-tumor activity using a mouse B16 melanoma model.
Assessment of non-fucosylated murine antibody binding to murine Fcγ receptors: CHO cells stably expressing murine FcγRI or FcγRIV were incubated with increasing concentrations of fucosylated mIgG2a 1C10 or non-fucosylated mIgG2a 1C10 (mIgG2a SEA-1C10). Samples were washed and a saturating amount of PE-anti-mouse IgG was added and incubated with the samples on ice for thirty minutes. Samples were washed again and labeled cells were analyzed by flow cytometry.
Results: Binding of the surrogate anti-CD40 antibodies to Chinese hamster ovary (CHO) cells expressing the murine FcγRI or FcγRIV (the murine equivalent to human FcγRIII/CD16) was assessed. Results are show in
Assessment of non-fucosylated anti-CD40 antibodies in a murine tumor model: 250.0E+3 B16F10 melanoma cells were given subcutaneously to C57BL/6 mice. Mice were randomized into cohorts each with tumor size of approximately 50 mm3 on average. Mice were then given interperitoneal injections of either an isotype control (mIgG2a), fucosylated mIgG2a 1C10, or non-fucosylated mIgG2a 1C10 (mIgG2a SEA 1C10), every other day for a total of three doses. Mice were monitored until the tumor size reached 1000 mm3, at which point the mice were sacrificed.
Results: The B16F10 sygeneic melanoma model was used to assess the in vivo efficacy of our non-fucosylated anti-CD40 antibody surrogates. C57BL/6 mice were implanted with B16F10 melanoma cells, and then treated with either mIgG2a isotype control, fucosylated mIgG2a 1C10, or non-fucosylated mIgG2a 1C10 (mIgG2a SEA 1C10). Tumor burden was monitored and mice were sacrificed when the tumor size reached 1000 mm3. Results are shown in
SEA-CD40 activity was compared to a related fucosylated antibody and to a fully agonistic anti-CD40 antibody, clone 21.4.1. Antibody 21.4.1 is a human anti-CD40 IgG2k agonistic antibody that is the parent clone of CP-870,893, an antibody that is currently being tested in a clinical trial of solid tumors in combination with PDL1. For amino acid sequence information for antibody 21.4.1, see, e.g., U.S. Pat. No. 7,338,660, which is herein incorporated for all purposes. Three functional areas were tested for the antibodies: ability to drive human B-cells differentiation, activation, and depletion, ability to activate primary human PBMC cultures, and ability to drive an antigen specific response.
Assessment of B-cell activation by anti-CD-40 antibodies: Experiments were performed using purified B-cells from fresh human whole blood or human peripheral blood mononuclear cells (PBMCs). B-cells were isolated from fresh human whole blood using ROSETTESEP™ isolation kit. The isolated, purified B-cells were cultured with increasing concentrations of SEA-CD40, antibody 21.4.1, or hexameric CD40 ligand, ENZO LIFE SCIENCES™ (10, 1, 0.1, 0.01, or 0.001 μg/mL) for 24 hours. B-cell activation was assessed as upregulation of CD80 by Flow Cytometry.
PBMCs were isolated as described above and were then cultured with increasing concentrations of SEA-CD40, antibody 21.4.1, or hexameric CD40L (10, 1, 0.1, 0.01, or 0.001 μg/mL) for 24 hours. The total number of B-cells assessed with CD19 staining assessed by flow cytometry.
Results: SEA-CD40 immune modulatory activity is dependent on the Fc portion of the antibody and its interaction with the CD16, the FcγRIII receptor. Results are shown in FIG. 17. SEA-CD40 does not induce B-cell activation in purified B-cells cultures which lack cells that express the Fcγ receptors needed for crosslinking of SEA-CD40. This differs from the CD40 activating antibody 21.4.1 which is able to drive B-cell activation in pure B-cell cultures similar to CD40 ligand.
PBMCs include cells that express the Fcγ receptors. Results for that cell population are shown in
Assessment of monocyte/macrophage activation by anti-CD-40 antibodies: Human PBMC cultures were isolated as described above. PBMC cultures were stimulated with increasing concentrations (0.0, 0.001, 0.01, 0.1, 1.0, or 10 μg/mL) of SEA-CD40, dacetuzumab, antibody 21.4.1, or an SEA-isotype control for twenty-four hours. Upregulation of CD80 is a marker of monocyte maturation. Surface expression of CD80 was assessed by flow cytometry.
Results: Results are shown in
Assessment of cytokine induction by anti-CD40 antibodies: Human PBMC cultures were isolated as described above. PBMC cultures were stimulated with increasing concentrations (0.0, 0.001, 0.01, 0.1, 1.0, or 10 μg/mL) of SEA-CD40, dacetuzumab, antibody 21.4.1, or an SEA-isotype control for twenty-four hours. Following stimulation, tissue culture supernatants were collected and inflammatory cytokines assessed by multiplexed LUMINEX™ analysis.
Results: Results are shown in
Assessment of T-cell induction by anti-CD40 antibodies: Human PBMC cultures were isolated as described above. 1×106 PBMCs were cultured in DMEM+10% FBS and incubated with 5 ug of M1 flu peptide, and with increasing concentrations (0.0, 0.001, 0.01, 0.1, 1.0, or 10 μg/mL) of SEA-CD40, dacetuzumab, or antibody 21.4.1 for five days. Cells and cell culture supernatants were then collected. IFN-γ levels were assessed in supernatants. Flu antigen-specific T-cells assessed by tetramer staining using by flow cytometry. Percent T-regulatory cells, a CD4+, CD25+, CD127 low population of cells was determined using flow cytometry.
Results: Results are shown in
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
This application is a Divisional Application of U.S. application Ser. No. 17/535,076, filed on Nov. 24, 2021, which is a divisional of U.S. application Ser. No. 15/522,614, filed on Apr. 27, 2017, which is a U.S. national stage filing under 35 U.S.C. § 371 of International Application No. PCT/US2015/058108, filed Oct. 29, 2015, which claims the benefit of U.S. Provisional Patent Application No. 62/072,031, filed Oct. 29, 2014 and U.S. Provisional Patent Application No. 62/134,955, filed on Mar. 18, 2015, each of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62134955 | Mar 2015 | US | |
| 62072031 | Oct 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17535076 | Nov 2021 | US |
| Child | 17734541 | US | |
| Parent | 15522614 | Apr 2017 | US |
| Child | 17535076 | US |
